1 Optimization of a generic drug product formulation using chemometric tools Marisa Rodrigues 1, 2 , Sandra Lopes 1 , José Menezes 2 , Paulo Salústio 3 , Maria Teresa Malta 1 , António M. Costa 1 1 Laboratórios Atral S.A. 2 Instituto Superior Técnico 3 Faculdade de Farmácia da Universidade de Lisboa ABSTRACT This study presents the pharmaceutical, analytical and manufacturing process development of a generic drug product, corresponding to the combination of a beta-lactam antibiotic and clavulanic acid in an extemporaneous oral suspension. In order to optimize the physical, chemical and pharmacotechnical properties of the generic drug product, a central composite face-centered (CCF) design with 17 experiments and three levels was applied. The critical factors of the formulation (flavour, suspending agent 1 and suspending agent 2) and its optimal ranges were estimated. The aim was to achieve a formulation with the following parameters: pH after reconstitution equal to the pH of the reference drug product, pH variation in ten days less than 1.0 pH unit, viscosity similar to the reference drug product and acceptable organoleptic characteristics. The relationship between the factors and responses were analysed using multiple linear regression and PLS models and the optimum point of the tested factors was determined: 82.82mg of suspending agent 1, 600mg of suspending agent 2 and 620mg of flavour. The implementation of experimental design tools and multivariate analysis proved to be useful for the formulation development of a generic drug product, reducing the time and resources spent, in comparison to an univariate approach. In order to select the final formulation of the generic drug product, the optimized formulation, the formulation initially suggested by references and the reference drug product were analysed. Finally, the analytical methods for the evaluation of the final formulation of the generic drug product and its manufacturing process were validated. Keywords: beta-lactam antibiotic, clavulanic acid, generic drug product, design of experiments (DoE), central composite face- centered (CCF), analytical methods validation, process validation. INTRODUCTION Suspension is a dispersion of finely insoluble particles (dispersed phase) in a liquid medium (continuous phase), usually aqueous. An extemporaneous oral suspension is a pharmaceutical form which ensures a high availability of drugs through oral absorption. [1] [2] The combination of a beta-lactam antibiotic and clavulanic acid is indicated in the treatment of polymicrobial infections caused by aerobic gram-positive, gram-negative and anaerobic due to the beta-lactam ring of beta-lactam antibiotics and the ability of clavulanic acid to inactivate beta-lactamase enzymes. [3][4] Drug product development is a creative and multidisciplinary process that turns a technological innovation and a market opportunity in products with economic profitability for the company. [5] In the development of a generic oral suspension, as the active substances are absorbed before being released into the bloodstream, the applicant does not need to do pre- clinical and clinical studies if he can demonstrate the similarity of the active substances release profile between generic and reference drug product by bioequivalence study. [6]-[9] The drug product under development and the respective reference product have the same dosage form: powder for extemporaneous oral suspension. The reconstituted suspension is a multiple dosage form medicine, which can be administered for 10 days, during which it maintains the biological activity for the intended use. Selection of components for the new formulation was based on literature review and analysis of the reference product. The qualitative composition chosen for the generic drug product is similar to the reference medicine, which corresponds to a beta-lactam antibiotic, clavulanic acid, suspending agent 1, suspending agent 2, glidant, sweetener, flavour and diluent. The experimental aim of this work is to optimize the formulation of a generic drug product. A central composite face-centered design (CCF) was used to study three factors of the formulation at three levels. CCF is a central composite design where the points of the star are placed in the centre of each face of the factorial space, requiring three levels for each factor. A central composite design is useful to study 2 to 5 factors, since a number greater than 6 implies an excessive number of experiments. This design is suitable for industrial pilot projects as it explores a large volume of experimental region with a reduced number of experiences factor. [10]- [12] This design includes eight experiments that represent a 2 3 factorial design (2 k ), plus 6 experiments (2k) representing the centre of each face of the factors space and 3 experiments of the centre point. [10]-[12] OBJECTIVE The goals of this study are: - Optimization of a generic drug product formulation using experimental design; - Definition and validation of the analytical methods; - Definition and validation of the manufacturing process; EQUIPMENT, MATERIAL AND METHODS Equipment Scale (AG204/MS105DU Mettler-Toledo), ultrasonic bath (Sonorex RK100 Bandelin), dissolution equipment (DT600HH Erweka). pH meter (744 Meter Metrohm), viscometer (LVT Brookfield), Karl Fischer (890 Titrando and 803 Ti Stand Metrohm) and HPLC Agilent 1100/1260 Series. Methods Karl Fischer - based on Ph. Eur. 8th Edition, chapter 2.5.12 Water: semi-micro determination. [13] High-resolution chromatographic method (isocratic) with UV detector for the identification and quantification of active substances. High-resolution chromatographic method (gradient) with UV detector - for the quantification of the beta-lactam impurities and degradation products content.
Transcript
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Optimization of a generic drug product formulation using chemometric tools Marisa Rodrigues1, 2, Sandra Lopes1, José Menezes2, Paulo Salústio3, Maria Teresa Malta1, António M. Costa1 1 Laboratórios Atral S.A. 2 Instituto Superior Técnico 3 Faculdade de Farmácia da Universidade de Lisboa
ABSTRACT
This study presents the pharmaceutical, analytical and manufacturing process development of a generic drug product, corresponding to the combination of a beta-lactam antibiotic and clavulanic acid in an extemporaneous oral suspension. In order to optimize the physical, chemical and pharmacotechnical properties of the generic drug product, a central composite face-centered (CCF) design with 17 experiments and three levels was applied. The critical factors of the formulation (flavour, suspending agent 1 and suspending agent 2) and its optimal ranges were estimated. The aim was to achieve a formulation with the following parameters: pH after reconstitution equal to the pH of the reference drug product, pH variation in ten days less than 1.0 pH unit, viscosity similar to the reference drug product and acceptable organoleptic characteristics. The relationship between the factors and responses were analysed using multiple linear regression and PLS models and the optimum point of the tested factors was determined: 82.82mg of suspending agent 1, 600mg of suspending agent 2 and 620mg of flavour. The implementation of experimental design tools and multivariate analysis proved to be useful for the formulation development of a generic drug product, reducing the time and resources spent, in comparison to an univariate approach. In order to select the final formulation of the generic drug product, the optimized formulation, the formulation initially suggested by references and the reference drug product were analysed. Finally, the analytical methods for the evaluation of the final formulation of the generic drug product and its manufacturing process were validated. Keywords: beta-lactam antibiotic, clavulanic acid, generic drug product, design of experiments (DoE), central composite face-centered (CCF), analytical methods validation, process validation.
INTRODUCTION
Suspension is a dispersion of finely insoluble particles (dispersed phase) in a liquid medium (continuous phase), usually aqueous. An extemporaneous oral suspension is a pharmaceutical form which ensures a high availability of drugs through oral absorption. [1] [2] The combination of a beta-lactam antibiotic and clavulanic acid is indicated in the treatment of polymicrobial infections caused by aerobic gram-positive, gram-negative and anaerobic due to the beta-lactam ring of beta-lactam antibiotics and the ability of clavulanic acid to inactivate beta-lactamase enzymes. [3][4] Drug product development is a creative and multidisciplinary process that turns a technological innovation and a market opportunity in products with economic profitability for the company. [5] In the development of a generic oral suspension, as the active substances are absorbed before being released into the bloodstream, the applicant does not need to do pre-clinical and clinical studies if he can demonstrate the similarity of the active substances release profile between generic and reference drug product by bioequivalence study. [6]-[9] The drug product under development and the respective reference product have the same dosage form: powder for extemporaneous oral suspension. The reconstituted suspension is a multiple dosage form medicine, which can be administered for 10 days, during which it maintains the biological activity for the intended use. Selection of components for the new formulation was based on literature review and analysis of the reference product. The qualitative composition chosen for the generic drug product is similar to the reference medicine, which corresponds to a beta-lactam antibiotic, clavulanic acid, suspending agent 1, suspending agent 2, glidant, sweetener, flavour and diluent. The experimental aim of this work is to optimize the formulation of a generic drug product. A central composite face-centered design (CCF) was used to study three factors of the formulation at three levels. CCF is a central composite design where the points of the star are placed in the centre of each face of the factorial space, requiring three levels for each factor. A central
composite design is useful to study 2 to 5 factors, since a number greater than 6 implies an excessive number of experiments. This design is suitable for industrial pilot projects as it explores a large volume of experimental region with a reduced number of experiences factor. [10]-[12] This design includes eight experiments that represent a 23 factorial design (2k), plus 6 experiments (2k) representing the centre of each face of the factors space and 3 experiments of the centre point. [10]-[12]
OBJECTIVE
The goals of this study are: - Optimization of a generic drug product formulation using experimental design; - Definition and validation of the analytical methods; - Definition and validation of the manufacturing process;
EQUIPMENT, MATERIAL AND METHODS
Equipment
Scale (AG204/MS105DU Mettler-Toledo), ultrasonic bath (Sonorex RK100 Bandelin), dissolution equipment (DT600HH Erweka). pH meter (744 Meter Metrohm), viscometer (LVT Brookfield), Karl Fischer (890 Titrando and 803 Ti Stand Metrohm) and HPLC Agilent 1100/1260 Series. Methods
Karl Fischer - based on Ph. Eur. 8th Edition, chapter 2.5.12 Water: semi-micro determination. [13] High-resolution chromatographic method (isocratic) with UV detector for the identification and quantification of active substances. High-resolution chromatographic method (gradient) with UV detector - for the quantification of the beta-lactam impurities and degradation products content.
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Viscosity - based Ph. Eur. 8th Edition chapter 2.2.8 Viscosity. [13] pH determination - based on Ph. Eur. 8th Edition chapter 2.2.3 Potentiometric determination of pH. [13] Mass uniformity - based on Ph. Eur. 8th Edition chapter 2.9.5 "Uniformity of mass. [13] Deliverable volume - based on USP chapter 698 Deliverable volume. [14]
Materials
Water (HPLC Milli-Q system), anhydrous monobasic sodium phosphate - NaH2PO4 (VWR), Methanol HPLC grade (Prolabo), Karl Fischer reagent (Panreac). Software
MODDE 6.0 da Umetrics AB. EXPERIMENTAL
Generic drug product
The initial proposal for qualitative and quantitative composition of the generic drug product was defined based on literature review (Table 1). Table 1: Qualitative and quantitative composition of the generic drug product.
Qualitative composition Quantitate composition
per flask (mg/100ml) Beta-lactam antibiotic Confidential
Diluted clavulanate Confidential Suspending agent 1 20 to 200
Sweetener Confidential Glidant Confidential
Suspending agent 2 200 to 1200 Flavour 420 to 620
Diluent (Filler) until 20,200
Based on this proposal, several formulations were prepared using different amounts of factors, along with fixed amounts of other components. Since formulation weight should be constant, the filler content is adjusted so, even with the variation of factors content, the sum of all the contents is always equal to 20,200mg. Drug and the excipients were homogeneously blended and subsequently conditioned in glass type II flask (20,200mg per flask). On MODDE the sum of the content of all components is equal to 1, which means that the components cannot be manipulated independently from each other and the content of each component must be between 0 and 1. [10] Experimental design
A CCF design was employed to study the influence of the amount of suspending agent 1 (X1), suspending agent 2 (X2) and flavour (X3) on the responses. The central point (0.0) was studied in triplicate. The factors and its levels were selected based on preliminary literature review. All other formulation variables were kept constant during the study. A total of 17 experimental runs were required to analyse the effect of each individual factor and their interaction in formulation characteristics in order to to optimize them. Table 2 summarizes the factors combinations and its levels.
Table 2: Experimental design runs, factor combination and levels. Run X1 X2 X3 Filler
* Central point (0.0) in triplicate. The dissolution profile similarity factor (Y1), variation of beta-lactam and clavulanic acid content in ten days (Y2 and Y3), pH after suspension reconstitution (Y4), variation of pH in ten days (Y5) and viscosity (Y6) were taken as the responses. Table 3 shows the response values obtained from the experimental design runs.
Table 3: Responses obtained on formulations evaluation. Run Y1 Y2 Y3 Y4 Y5 Y6
*Knowing that the initial pH is higher than 5.0 pH units, a percentage of 100% is attributed to 5.0 pH units, while 35.7% is equivalent to 14 pH units. **As the ideal is not occur pH changes during the 10 days, a percentage of 100% is attributed to 0.1 pH units, while 2.0 pH units is equivalent to 5%.
RESULTS AND DISCUSSION
Optimization of the generic drug product formulation
using a composite face-centered design
Table 4 summarizes the characteristics of the multivariate models applied for optimization of the responses. f2 has not been studied as a model response since all of the 17 formulations have a dissolution profile similar to the reference product (percentage dissolved ≥ 85% at 15 minutes). The variation of beta-lactam antibiotic content has also not been studied because no significant differences among the 17 experiments were observed.
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Table 4: Summary of the multivariate models applied for optimization of the responses. Model Response Model type Fit parameters Model adjustment ANOVA
1 pH at time zero MLR
R2: 0.96 Q2: 0.91 RSD: 0.68 N: 17 runs
Outliers: Not found. Significant coefficients: suspending agent 2
p regression: 0.000 p Lack of fit: 0.907
2 pH variation over 10 days
MLR
R2: 0.86 Q2: 0.75 RSD: 0.54 N: 17 runs
Outliers: Not found. Significant coefficients: suspending agent 2
p regression: 0.000 p Lack of fit: 0.888
3 Viscosity MLR
R2: 0.95 Q2: 0.87 RSD: 297.17 N: 16 runs
Outliers: run 12. Significant coefficients: suspending agent 1 and suspending agent 2
p regression: 0.000 p Lack of fit: 0.132
4 Variation of clavulanic acid content over 10 days
MLR
R2: 0.90 Q2: 0.62 RSD: 0.40 N: 16 runs
Outliers: run 10. Significant coefficients: suspending agent 2 and flavour
p regression: 0.002 p Lack of fit: 0.823
Identification of statistically significant factors effects and its optimal ranges
a) pH at time zero
pH at time zero is influenced by suspending agent 2. A
lower content of this component contributes to a pH after
reconstitution nearest to 5.0 pH units (Figure 1).
According to literature review, pH after reconstitution should be between 5.0 and 5.5. In Figure 2, the target region is identified as the region corresponding to a content of suspending agent 2 between 909 and 202mg per 100ml (ratio from 0.01 to 0.045), regardless suspending agent 1 or flavour content.
Figure 1: Influence of suspending agent 2 on pH at time zero.
Figure 2: Optimal ranges of suspending agent 2 – model #1 (initial pH).
b) pH variation over 10 days
The pH variation is influenced by suspending agent 2. A
higher concentration of this component contributes to a
lower reduction of pH during the 10 days of the
reconstituted suspension storage (Figure 3).
According to literature review, pH should not decrease more than 1 pH unit over ten days, preferably no more than 0.8 pH units. Figure 13 shows the target region as the region corresponding to a content of suspending agent 2 between 707 and 1212mg per 100ml (ratio between 0.035 and 0.060) and a content of suspending agent 1 between 20 and 162mg (ratio between 0.001 and 0.008) (Figure 4).
Figure 3: Influence of suspending agent 2 on pH variation.
Figure 4: Optimal ranges of suspending agent 2 – model #2 (pH decrease)
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c) Viscosity
Viscosity is influenced by suspending agent 1 and 2. A
higher content of these components contributes to a higher
viscosity value (Figure 5).
The reference drug product presents 610 cPs. The target viscosity value was defined as 610 ± 61 cPs. According to Figure 6, an optimum viscosity value is a function between the two suspending agents. A higher content of a suspending agent implies a lower content of the other, due to their synergistic effect on viscosity value.
Figure 5: Influence of suspending agent 1 and 2 on viscosity.
Figure 6: Optimal ranges of suspending agents 1 and 2 – model #3
(viscosity).
d) Clavulanic acid degradation over 10 days
Clavulanic acid degradation is influenced by suspending agent 2 and flavour. A content of suspending agent 2 between 505mg and 808mg contributes to a smaller reduction of clavulanic acid content in 10 days and a lower concentration of flavour contributes to a minor degradation in 10 days (Figure 8). Clavulanic acid degradation should be the less than 15% (clavulanic acid overage) to ensure the therapeutic effect of the generic drug product over the ten days of usage. According to Figure 8, in the presence of 420/520mg of flavour, the clavulanic acid degradation is less than 15%, with any content of suspending agents (within the operating range). With 420mg of flavour, the clavulanic acid degradation could be lower than 13.5%, with 505mg (ratio 0.025) or more of suspending agent 2 and 90.9mg (0.0045 ratio) or less of suspending agent 1. With 520mg of flavour, the lowest clavulanic acid degradation that could be obtained is 14%, if the content of suspending agent 2 is less than 505mg (ratio of 0.025). In the presence of 620mg of flavour, the clavulanic acid degradation is less than 15% with a content of suspending agent 2 lower than 808mg (ratio 0.040) and a content of suspending agent 1 between 60.6 and 202mg (0.003 to 0.01 ratio).
Figure 7: Influence of suspending agent 2 and flavour on clavulanic acid degradation.
Figure 8: Optimal ranges of suspending agent 2 and flavour – model #4 (clavulanic acid degradation)
Optimal formulation
The aim is to achieve a formulation with the following parameters: pH after reconstitution equal to reference drug product (5.30), pH variation in ten days less than 1.0 pH unit and viscosity similar to the reference drug product (610cPs ± 10%). 1. According to model #1, to obtain a pH after reconstitution equal to 5.30 (94.34%), the content of suspending agent 2 must be between 268.7 and 606mg and the content of suspending agent 1 between 20 and 200 mg.
2. The content of suspending agent 2 was fixed at 600mg in order to approximate the content between generic and reference drug product. With 600mg of suspending agent 2 it is possible:
• pH minimum value of 5.29 (94.5%) at time zero, for 200 mg of suspending agent 1 and 620mg of flavour; • pH maximum value of 5.43 (92.1%) at time zero, for 20mg suspending agent 1 and 520mg flavour. 3. To fulfil the requirement for viscosity value (561-670cPs), a formulation with 600 mg of suspending agent 2 must have a content of suspending agent 1 between 82.01 and 95.75mg. 4. According to MODDE optimization tools, in the presence of 600mg of suspending agent 2 and 82.82 to 95.75mg of suspending agent 1, the optimal pH value corresponds to 5.31 (94.1%), with 620mg of flavour (Figure 9).
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Figure 9: Optimal formulation.
Therefore, optimal generic drug product formulation is obtained with 82.82mg suspending agent 1, 600mg suspending agent 2 and 620mg flavour. The following experimental results are predicted (with software MODDE) for this formulation: • pH at time zero = 5.32 (94.07%) • Viscosity = 569 cps • pH variation in 10 days = 1.02 (9.81%) • Clavulanic acid degradation in 10 days = 14.61%.
Definition of the generic drug product formulation
Two formulations (Table 5) were compared to reference drug product, based on analysis of organoleptic characteristics, pH, viscosity and clavulanic acid degradation: 1. Formulation obtained from literature review (batch DG15038); 2. Formulation determined using DoE (batch DG15039); 3. Reference drug product. Table 5: Formulations of the batches DG15038 e DG15039.
Table 6 shows the results obtained. The formulation obtained from literature review (DG15038) is more appreciated as it presents a fruit smell, sweet taste and a nice mouthfeel. The formulation determined using DoE (DG15039) presents a fruit smell and a sweet taste, but with an unpleasant, sandy texture. This texture is probably caused by suspending agent 1, present in a larger quantity than the formulation obtained from literature review (more 25mg per 100ml).
pH analysis (pH after reconstitution and after 10 days of storage) showed similar pH between both formulations and reference drug product, within the range of 5.0 to 5.5 pH units. The range of 555 to 679 cPs (610 cPs ± 10%) had been considered as the optimum viscosity value and only the formulation determined with DoE results complies with this criteria. Clavulanic acid therapeutic activity is expected to be preserved over 10 days with both formulations tested, with a content higher than 100% after 10 days of storage. Table 6: Formulation evaluation results.
Test DG15038 DG15039 Reference
drug product Organoleptic characteristics
3 2 2
pH at time zero (pH units)
5.41 5.44 5.39
Increase of pH over 10 days (pH units)
0.91 0.85 0.78
Decrease of clavulanic acid after 10 days (%)
13.0 12.3 13.0
Viscosity (cPs) 463 603 617
Table 7 summarizes the benefit of each formulation. Since organoleptic characteristics were considered more relevant than viscosity, the formulation obtained from literature review was selected as the future formulation of the generic drug product. Table 7: Selection of the generic drug product.
Test DG15038 DG15039
Organoleptic characteristics ✓ ✗
pH at time zero (pH units) ✓ ✓
Variation of pH over 10 days (pH units)
✓ ✓
Variation of clavulanic acid over m 10 days (%)
✓ ✓
Viscosity (cPs) ✗ ✓
Definition and validation of analytical methods
The analytical methods were developed and defined based on literature review, including active substances and finished product monographs present on pharmacopoeias. Dissolution, assay and quantification of impurities methods were validated. Table 8, 9 and 10 show the acceptance criteria and the results obtained for each validation parameter. As the results obtained comply with their acceptance criteria, it could be concluded that these analytical methods are suitable for their intended use: quantification of active substances (beta-lactam antibiotic and clavulanic acid)/impurities in the finished product of the generic powder for oral suspension, in the defined work range.
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Table 8: Quantification of active substances after dissolution test. Validation parameter Acceptance criteria Results
Specificity Presence of solvent, placebo or impurities should not interfere with the active substances signal (interference ≤ 0.5% and resolution ≥ 1.5)
Solvent and placebo do not interfere with the active substances analytical signal (absence of co-eluting and resolution factor ≥ 1.5 for all peaks)
Accuracy Average recovery: 95.0-105.0% Average recovery 1/2 = 100.4/103.8% RSD 1/2 = 0.13/0.20%
System precision RSD ≤ 1% RSD area 1 = 0.05% RSD area 2 = 0.25%
Analytical solutions stability Variation ≤ 5% Variation < 2% Work range ± 20% of specified range 20-120% of target concentration
1 – Beta lactam antibiotic; 2 – Clavulanic acid
Table 9: Assay of active substances.
Validation parameter Acceptance criteria Results
Specificity Presence of solvent, placebo or impurities should not interfere with the active substances signal (interference ≤ 0.5% and resolution ≥ 1.5)
Solvent and placebo do not interfere with the active substances analytical signal (absence of co-eluting and resolution factor ≥ 1.5 for all peaks)
Accuracy Average recovery: 98.0-102.0% Individual recovery: 97.0-103.0%
Average recovery 1/2 = 100.1/99.7% RSD 1/2 = 0.54/0.53%
System precision RSD ≤ 1% RSD area 1 = 0.18% RSD area 2 = 0.19%
Analytical solutions stability Variation ≤ 2% Variation < 1% Work range 98-102% 70-130% of target concentration
1 – Beta lactam antibiotic; 2 – Clavulanic acid
Table 10: Quantification of beta-lactam antibiotic impurities.
Validation parameter Acceptance criteria Results
Specificity Presence of solvent. placebo or impurities should not interfere with the active substances signal (interference ≤ 0.5% and resolution ≥ 1.5)
Solvent and placebo do not interfere with the active substances analytical signal (absence of co-eluting and resolution factor ≥ 1.5 for all peaks)
Accuracy
Impurity content RSD (%)
Average recovery =104.3% RSD =12.5 %
X ≤ 0.1% 50.0-150.0% 0.1% < X ≤ 0.5% 70.0-130.0% 0.5% < X ≤ 1.0% 80.0-120.0%
X > 1.0% 90.0-110.0%
System precision
Impurity content RSD (%)
RSD area = 0.38% X ≤ 0.1% ≤ 10.0 0.1% < X ≤ 0.5% ≤ 7.5 0.5% < X ≤ 1.0% ≤ 5.0 X > 1.0% ≤ 3.0
LOQ Lowest concentration (equal or below to reporting threshold) RSD ≤ 10.0%
Individual recovery: 110-130% Average recovery = 118.1% RSD = 6.3%
Analytical solutions stability
Variation ≤ 10% Variation of impurities sum < 10% (stored at ambient temperature with exposure to light for 48 hours)
Work range Reporting threshold to 120% of specification Reporting threshold to 120% of specification level 1 – Beta lactam antibiotic; 2 – Clavulanic acid
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Definition and validation of the manufacturing process
The manufacturing process was defined according to the characteristics of the active substances and the pharmaceutical form in which they will be marketed. The manufacturing process of the generic drug product, to be marketed as a powder for extemporaneous oral suspension, is divided into two stages: 1. Preparation: weighing, sieving and blend of raw materials; 2. Filling: filling bottles with the pre-defined amount of mixture. The validation of this manufacturing process included: Blend validation: the mixture homogeneity was checked in 10 points of the blender at three different blending times (10, 20 and 30 minutes); Filling validation: the product quality was checked in three locations (beginning, middle and end of the filling line); Evaluation of the quality of the final product. In this project only two batches were analysed up to now, but the analysis of the third batch (biobatch) will be performed to complete the process validation. Table 10 reports the process validation tests up to now and their specifications for the physical-chemical analysis of the intermediate and finished product Table 10: Specifications of process validation tests.
Test Specification Description (visual) White or almost white powder Identification (HPLC) Positive Water content (Karl-Fischer)
≤ 12.0%
pH of reconstituted suspension (potentiometry)
3.8 – 6.6
Ressuspendibility (visual)
Homogeneous, white or slightly yellow with fruit smell suspension
Microbial contamination Total aerobic ≤ 103 CFU/ml Fungi and yeasts ≤ 102 CFU/ml E. coli – Absent/ml
* After completion of reference product stability studies
Blend process validation
Blend validation aims to prove the powder homogeneity at the defined blend time (30 minutes), by checking the active substances content at different sampling points of the blender. The homogeneity was also checked at 10 and 20 minutes to ascertain if the blend time could be reduced. The average contents obtained for beta-lactam antibiotic and clavulanic acid are presented in Table 11. According to specifications, relative standard deviation (RSD) between samples from different points of the blender should be, no more than 5%. It is noted that the RSD is less than 5% at any blend time for both batches. Therefore, the powder mixture is considered homogeneous after 10 minutes blending.
Table 11: Results of blend process validation.
Batch Time (min)
API Average
(%) RSD (%)
1
10 beta-lactam antibiotic 114.2 1.3
clavulanic acid 129.1 4.2
20 beta-lactam antibiotic 115.9 0.3
clavulanic acid 127.8 0.8
30 beta-lactam antibiotic 116.6 0.7
clavulanic acid 126.9 0.5
2
10 beta-lactam antibiotic 115.0 0.5
clavulanic acid 127.6 1.1
20 beta-lactam antibiotic 113.9 1.5
clavulanic acid 124.4 1.9
30 beta-lactam antibiotic 116.6 0.7
clavulanic acid 127.1 0.6
Filling process validation
Filling process validation ensures the product remains constant throughout the packing line, in accordance with the desired quality specifications. The results are shown in Table 12. Table 12: Results of filling process validation (two batches).
Product description and identification of active substances are consistent between batches and meet the acceptance criteria. Average pH of reconstituted suspension (5.1) is included in the specified range (3.8 to 6.6 pH units). Active substances assay revealed average contents of 113.9% and 124.8% for beta-lactam antibiotic and clavulanic acid, respectively. These results are within the acceptance criteria (content 95.0 to 120.0% for beta-lactam antibiotic and 95.0 to 130.0% for clavulanic acid and RSD ≤ 5%). Water content should be less than 12.0% therefore, both batches meet the specification. The average mass of 10 bottles of powder for oral suspension did not differ more than 5% of the desired mass (22.22g), which is in conformity with the specification.
Analysis of the final product The analysis of the final product must prove the agreement between its characteristics and the predefined specifications. Table 13 shows the results obtained. The results of product description, identification of active substances, ressuspendibility and microbial contamination are consistent between batches and meet the acceptance criteria. Average pH of reconstituted suspension (5.1) is included in the specified range (3.8 to 6.6 pH units). Water content should be less than 12.0% therefore, both batches meet the specification. Assay of active substances revealed average contents of 113.7% and 125.6% for beta-lactam antibiotic and clavulanic acid, with a RSD of 0.7 and 0.2% respectively.
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These results are within the respective acceptance criteria (content 95.0 to 120.0% for beta-lactam antibiotic and 95.0 to 130.0% for clavulanic acid and RSD ≤ 5%). Impurities total content is identical between both batches. Dissolution test shows complete dissolution of both actives substance after 15 minutes, which meet their acceptance criteria (content dissolved ≥ 85% after 15 minutes). The extemporaneous oral suspension must have a minimum volume of 95ml so, 106ml of available volume fulfils this criteria. The average mass of 10 bottles of powder for oral suspension did not differ more than 5% of the desired mass (22.22g), which is in conformity with the specified criteria. Table 14: Results of the final product analysis.
Test Batch
1 2 Description White powder Identification Positive
Water content (%) 10.55 10.40
pH 5.12 5.15
Resuspend White and homogeneous suspension with a fruit
smell Assay (%) - Beta-lactam - Clavulanic acid
113.1 125.4
114.2 125.7
Related impurities (%): -Impurity 1 - Impurity 2 - Impurity 3 - Impurity 4 - Total
0.19 0.18 0.42 0.40 1.19
0.22 0.18 0.41 0.39 1.21
Dissolution in 15 minutes (%): - Beta-lactam - Clavulanic acid
113.7 123.6
112.4 121.5
Available volume (ml) 106 106 Mass uniformity - Average (g) - RSD (%)
22.06 0.61
22.18 0.61
Microbial contamination Complies
CONCLUSION
A generic formulation, based on literature review, was optimized in order to display chemical and pharmacotechnical properties similar to its reference drug product (using a different flavour). A CCF design made it possible to understand the influence of the factors flavour, suspending agent 1 and suspending agent 2 content on the responses using only 17 experiments. After analysis and interpretation of the models, the optimum point of the tested factors was obtained: 82.82mg suspending agent 1, 600mg suspending agent 2 and 620mg flavour. According to Table 15, the results predicted with MODDE software are in agreement with the results observed, which confirms the validity of the developed models. Table 15: Results predicted versus experimental results.
Test Results
predicted with MODDE
Results observed (batch DG15039)
pH at time zero (pH units)
5.3 5.4
pH variation over 10 days (pH units)
1.0 0.9
Clavulanic acid variation over 10 days (%)
14.6 12.3
Viscosity (cPs) 569 603
Based on all mentioned criteria, the formulation suggested by literature review (batch DG15038) was selected as the future formulation for generic drug product. The partial process validation met the criteria specified on the three stages tested, therefore, the manufacturing process of the generic drug product is suitable for its purpose. The analytical methods for the quantification of active substances and impurities and quantification of the active substances after the dissolution test were also successfully validated. Future work
This project focused on the intermediate stages of a new generic drug product development, which will be followed by three future steps: ● Complete process validation (third batch analysis); ● Stability studies of the generic drug product (packed in their final packaging); ●Bioequivalence and bioavailability studies (determination of in vivo similarity between reference and generic drug product).
REFERENCES [1] Aulton, M. E. (2005). Pharmaceutics The Science of Dosage Form Design (2nd ed.). Churchill Livingstone. [2] Lachman, L.; Lieberman, H.; Kaning, J. (2001). Teoria e prática na indústria farmacêutica. Fundação Calouste Gulbenkian. [3] Suarez, C.; Gudiol, F. (2009). Antibióticos betalactámicos. Enfermedades Infecciosas y Microbiología Clínica 27(2), 116–129. [4] Prontuário terapêutico on-line. 1. Medicamentos anti-infecciosos. 1.1 Antibacterianos. INFARMED. Consultado em Agosto de 2015 em www.infarmed.pt/prontuario/index.php. [5] Patrício, M. (2014). Desenvolvimento Farmacêutico de medicamento: Área analítica. Universidade Lusófona de Humanidades e Tecnologias [6] Decreto-Lei n.º 176/2006, de 30 de Agosto. Estatuto do medicamento. Legislação Farmacêutica Compilada. [7] European Medicines Agency. (2012). Perguntas e respostas sobre os medicamentos genéricos )EMA/393905/2006 Rev. 2). Consultado em Agosto de 2015 em http://www.ema.europa.eu/docs/pt_PT/document_library/Medicine_QA/2009/11/WC500012382.pdf. [8] Ponciano, F. (2013). Preparação do processo de Autorização de Introdução no Mercado. Ordem dos farmacêuticos. Disponível em Consultado em Agosto de 2015 em www.ordemfarmaceuticos.pt/xFiles/scContentDeployer_pt/docs/Docs199.pdf. [9] Diretiva 2001/83/EC. Jornal Oficial das Comunidades Europeias. [10] Eriksson, L., Johansson, E., Kettaneh-Wold, N. Wikstrom, C., Wold, S. (2000) Design of experiments - Principles and Applications. Umetrics AB. [11] Sigh, B., Bhatowa, R., Bhushan, C., Kapil, R. (2011). Developing micro-/nanoparticulate drug delivery systems using “design of experiments”. Journal of Pharmaceutical Investigation, 1(2), 75-87. [12] NIST/SEMATECH e-Handbook of Statistical Methods. Consultado em Agosto de 2015 em http://www.itl.nist.gov/div898/handbook/pri/section3/pri3361.htm. [13] Farmacopeia Europeia (8th ed.). (2014). [14] United States Pharmacopeia and the National Formulary (USP38-NF33). (2015).
